The present invention relates to a method of and apparatus for freezing blood cells, bone marrow and other similar biological tissue, and more particularly to such a system and method wherein the tissue is heated to compensate for excessive cooling of the tissue by a refrigerating gas.
There is a medical need for the preservation of blood cells, bone marrow, and other biological tissue. Long-term storage in the frozen state has proven to be a successful method and has been practiced for some years. It has been known for some time that the viability (survivability) of frozen blood cells and biological tissue depends upon the rate at which such material is frozen, i.e., the rate at which the tissue is transferred from the liquid to the solid state. Cell destruction can occur if freezing takes place excessively fast or slow. While the addition of cryoprotective agents helps alleviate the situation and has proven successful for certain cell types, freezing rate is still considered to be a primary factor in cell viability.
The purpose of the invention is to provide a more precise apparatus for and method of controlling the freezing rate of biological tissue and thereby permit medical researchers to establish the optimum (maximum viability) freezing rate, thus increasing the yield of frozen cells and, consequently, reducing the cost thereof.
It is another object of the invention to enable the yield of frozen white blood cells, bone marrow and other similar biological tissue to be increased to the point where they would find wide acceptance in the treatment and cure of disease.
In the prior art, before the freezing process begins, the tissue to be frozen is mixed with a cryoprotective agent in a container, e.g., an ampule or sample bag. Typically, the container is then placed in a standard, commercially available biological freezer. The freezer uses a liquid nitrogen freezing solution which vaporizes to provide cooling gas to which heat is transferred from the tissue being frozen. The system is programmed to drop the cooling gas temperature at a desired rate; i.e., control is maintained over the cooling gas and not the freezing sample (although the temperature of the outside surface of the sample is monitored and recorded). After the sample is completely frozen, it is quickly transferred to a storage freezer where it is kept until needed.
The prior art systems invariably encountered problems in maintaining temperature control of the freezing solution while the latent heat of fusion was being released from the freezing solution. At the freezing point of the tissue, a large amount of heat (compared to the cooling of the liquid or solid) must be removed to accomplish the change of state from liquid to solid. In an ideal solution, this change of state occurs at a constant fluid-solid temperature for the cells.
A disadvantage of the prior art system is that temperature control is maintained of the cooling gas rather than of the sample being frozen. Variations in thermal properties and quantity being frozen from one sample to another cause widely varying sample freezing rates, which is unsatisfactory because cell viability is strongly dependent on freezing rate.
It is, therefore, a further object of the invention to provide a new and improved apparatus for and method of freezing biological tissue consistently, regardless of variations in the properties and the quantity of the tissue.
Modifications of the above described system provide some improvement. For example, the temperature of the tissue can control the flow of liquid nitrogen refrigerant to the freezing chamber. However, freezer gas temperature response is slow and it has been found that a uniform, repeatable freezing curve for the tissue cannot be maintained.
Another modification to the previous freezing system involves increasing the flow of liquid nitrogen refrigerant to the freezing chamber when the sample being frozen reaches the freezing point. In reality, the freezing liquid experiences a subcooling effect near the freezing point. The temperature of the sample drops below the freezing temperature until freezing starts and rises a few degrees when freezing is initiated. By observing the output of a thermocouple located on the sample, one can tell when freezing starts. At this point, the liquid nitrogen refrigerant can be turned on at full flow to provide maximum cooling. Once again, however, the response of the cooling gas temperature is not fast enough to maintain the required cooling.
Attempts at programming the freezer to provide more or less cooling at different phases of the freezing process are unsuccessful because: (1) the occurrence time of the phase change from liquid to solid can vary from sample to sample, and (2) the freezer cooling gas cannot change temperature quickly enough.